Tissue stain and use thereof

ABSTRACT

Provided is a tissue staining composition containing carbon particles having a mean particle diameter less than 0.3 μm in diameter (optionally less than 0.2 μm in diameter) together with one or more agents that maintain the carbon particles in suspension (for example, an anti-settling agent and/or surfactant). In certain circumstances, the anti-settling agent may also have mucoadhesive properties. The tissue staining composition is visually dark and does not disperse rapidly when introduced into regions of tissue of interest making it ideal for marking the regions that can be visualized clearly and over prolonged periods of time via, for example, direct visualization, endoscopic or laparoscopic inspection. The invention also provides methods of making and using the tissue staining composition for marking regions of tissue of interest, for example, gastrointestinal tissue, as well as other tissues.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of, and priority to, U.S.Provisional Patent Application Ser. No. 62/458,805 filed Feb. 14, 2017,which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The invention relates generally to a tissue staining composition formarking regions of living tissue, for example, regions of thegastrointestinal tract, in order to identify and visualize the regionsat a later point in time.

BACKGROUND

In certain medical procedures there is a need to mark regions of tissue(for example, cancerous or pre-cancerous lesions) so that they can thenbe later identified, for example, via endoscopic, laparoscopic, or opensurgical procedures, weeks, months or years later. Such markings canidentify tissue to be surgically excised via a subsequent procedure orpermit a physician or other medical provider to assess whethercytological or morphological changes have occurred over time that mayjustify medical intervention (for example, surgery or drug treatment) toremove or treat the tissue in question.

This type of marking and monitoring is routine in the field ofgastroenterology. For example, during an endoscopic examination of thegastrointestinal tract, a physician or other medical practitioner maywish to mark an abnormal area or potential lesion for subsequentmonitoring and/or surgical intervention. In a marking procedure, astaining composition is injected through the luminal mucosal surface ofthe gastrointestinal tract into the submucosal tissue. However, existingstains (e.g., methylene blue, indigo carmine, indocyanine green-basedstains) may be difficult to visualize endoscopically even at the time ofinjection into tissue of interest because the stain may leak or diffuseout of the tissue at the point of entry and thus visually obscure thearea being marked. Furthermore, even after injection, it may becomedifficult to identify the marked regions months to years later becausethe marks may fade or even disappear over time. In other words, suchstains may not facilitate permanent marking of tissue to accommodateaccurate, long term tracking of tissue of interest. As a result, it canbe difficult or even impossible for a physician or other medicalpractitioner to monitor with confidence abnormal tissue, pre-cancerousor cancerous lesions over time. There are similar needs for monitoringother tissues of interest, for example, in the bladder, urinary tract,breast, lymph nodes, central and peripheral nervous system, and lungs.

Furthermore, it has been observed that with certain tissue stains, thestaining agents, if particulate, may settle out of solution over time.As a result, the instructions for use typically require that the tissuestain be shaken or vortexed so that the staining agents are resuspendedor redistributed prior to use to ensure adequate marking. However, thisstep may not be performed adequately, if at all, prior to injection, andas a result differing amounts of staining agent may be delivered duringdifferent marking procedures or when different areas are marked duringthe same procedure, resulting in inconsistent marks or marks whereinsufficient staining agent has been introduced to create a durablemark.

Furthermore, certain stains (e.g., india ink) often contain impuritiessuch as shellacs, phenols, ammonia, and animal products. Theseimpurities can be associated with complications such as inflammatoryreactions or may even be carcinogens.

Despite the advances made in tissue marking agents, for example,endoscopic tissue marking agents, to date, there is still an ongoingneed for agents with improved marking reliability, durability, andsafety.

SUMMARY OF THE INVENTION

It has been discovered that it is possible to produce a biocompatible,carbon particle-based tissue staining composition that is easy andreliable to use, is easily observable during injection into tissue, andprovides a durable, reliable mark that can be visualized over time,either by direct observation or by inspection using an endoscope,laparoscope or other similar device. This has been facilitated by thediscovery that, when using carbon particles as the staining agent, thecarbon particles should have a mean particle diameter less than 0.3 μmin diameter (for example, less than 0.2 μm in diameter) to enhance thedarkness of the staining agent. Depending upon the presence of an agent,such as a surfactant, such as a non-ionic surfactant, present in anamount sufficient to reduce particle agglomeration, the particles mayremain in suspension for prolonged periods of time with little or nosedimentation (settling). However, the composition may also comprise ananti-settling agent and/or a mucoadhesive agent. Under certaincircumstances, a single agent can act as both an anti-settling agent anda mucoadhesive agent. The tissue staining compositions described hereinhave improved storage characteristics because the carbon particles areless likely to settle out of solution upon storage, provide the sameuniform optical density throughout a marking procedure, are darker thanother commercially available carbon particle-based tissue stains andthus are easier to visualize at the time of injection into a region oftissue of interest, and provide a durable mark at the region that can bemore readily visualized over time. In addition, the tissue stainingcomposition can be thermally sterilized, for example, by autoclaving,because the formulation of the tissue stain is heat stable.

In one aspect, the invention provides a liquid, tissue stainingcomposition comprising: carbon particles at a concentration of fromabout 0.025% to about 2.0% (w/v) having a mean particle diameter of lessthan 0.3 μm in diameter (e.g., less than 0.2 μm in diameter); anoptional anti-settling agent (ASA), for example, an ASA selected fromthe group consisting of hydroxyethyl cellulose, hydroxypropyl cellulose,dextran, and guar gum at a concentration of from about 0.025% to lessthan 5.0% (w/v) (for example, from about 0.05% to less than 5.0% (w/v),from about 0.05% to about 1.0% (w/v), from about 0.1% to about 1.0%(w/v), or from about 0.1% to about 0.5% (w/v)); and an optionalmucoadhesive agent.

In another aspect, the invention provides a liquid, tissue stainingcomposition comprising: carbon particles at a concentration of fromabout 0.025% to about 2.0% (w/v) having a mean particle diameter of lessthan 0.3 μm in diameter (e.g., less than 0.2 μm in diameter); ananti-settling agent (ASA), for example, an ASA selected from the groupconsisting of hydroxyethyl cellulose, hydroxypropyl cellulose, dextran,and guar gum at a concentration of from about 0.025% to less than 5.0%(w/v) (for example, from about 0.05% to less than 5.0% (w/v), from about0.05% to about 1.0% (w/v), from about 0.1% to about 1.0% (w/v), or fromabout 0.1% to about 0.5% (w/v)); and an optional mucoadhesive agent.

In certain embodiments, and with regard to each of the foregoingaspects, the carbon particles have a mean particle diameter less than0.2 μm, for example, from about 0.05 μm to less than 0.2 μm.Furthermore, in certain embodiments, the composition can becharacterized in that, when centrifuged for 60 minutes at 5,000×g, lessthan 50% of the carbon particles settle out of solution.

The carbon particles can be derived from carbon black, activated carbon,unactivated carbon or a combination thereof. The carbon particlespreferably have a level of polycyclic aromatic hydrocarbons of nogreater than 0.5 ppm based on the total amount of carbon particles.Preferably the carbon particles are depyrogenated or are otherwisepyrogen free. This can be achieved, for example, by heating carbonpowder at 220° C. for 1 hour to produce pyrogen free dry carbon.

In certain embodiments, the ASA is also a mucoadhesive agent (or hasmucoadhesive properties). Exemplary ASAs with mucoadhesive propertiesinclude, for example, hydroxyethyl cellulose, hydroxypropyl cellulose,dextran, and guar gum.

In another embodiment, the composition further comprises aviscosity-increasing agent. The viscosity-increasing agent can bepresent at a concentration of from about 5% to about 25% (w/v).Exemplary viscosity-increasing agents include, for example, glycerol,propylene glycol, isopropylene glycol, polyethylene glycol, celluloseand combinations thereof.

In another embodiment, the composition further comprises an anti-foamingagent. In certain embodiments, the anti-foaming agent can be present ata concentration of from about 0.005% to about 1.0% (w/v). Exemplaryanti-foaming agents include, for example, dimethicone and simethiconeand combinations thereof.

In another embodiment, the composition further comprises a surfactant,for example, a non-ionic surfactant. In certain embodiments, thesurfactant can be present at a concentration of from about 0.01% toabout 2.0% (w/v). Exemplary surfactants include, for example,pharmaceutically acceptable polyoxyethylene sorbitan esterified with afatty acid, for example, Tween® such as Tween® 80.

In another embodiment, the composition further comprises a preservative,for example, benzyl alcohol, methyl or ethyl paraben, and benzalkoniumchloride. In certain embodiments, the preservative can be present at aconcentration of from about 0.01% to about 4.0% (w/v).

In certain embodiments, the composition, for example, for use ingastrointestinal applications, is capable of passing through a 25 gaugeneedle about 240 cm in length upon application of a force no greaterthan about 32N (for example, from about 22 to 32N, or about 22N or less,or about 10N or less) to push the composition through the needle. Incertain other embodiments, the composition, for example, for use inbreast cancer tissue marking, is capable of passing through a 14 gaugeneedle about 5 cm in length upon application of a force no greater thanabout 32N (for example, from about 22 to 32N, or about 22N or less, orabout 10N or less) to push the composition through the needle.

In another embodiment, the composition comprises from about 0.025% toabout 2.0% (w/v) carbon particles or from about 0.1% to about 2.0% (w/v)carbon particles; optionally, and when present, from 0.025% to less than5.0% (w/v) of an ASA selected from the group consisting of hydroxyethylcellulose, hydroxypropyl cellulose, dextran, and guar gum; optionally,and when present, from about 5.0% to about 25% (w/v)viscosity-increasing agent; from about 0.005% to about 1.0% (w/v)anti-foaming agent; and from about 0.1% to about 2.0% (w/v) surfactant.In another embodiment, the composition comprises from about 0.025% toabout 1.0% (w/v) carbon particles or from about 0.1% to about 1.0% (w/v)carbon particles; optionally, and when present, from about 0.025% toabout 1.0% (w/v) of an ASA selected from the group consisting ofhydroxyethyl cellulose, hydroxypropyl cellulose, dextran, and guar gum;optionally, and when present, from about 12% to about 18% (w/v) of aviscosity-increasing agent; from about 0.05% to about 0.25% (w/v)anti-foaming agent; and from about 0.7% to about 1.5% (w/v) surfactant.Each of the foregoing compositions may further comprise a preservative,for example, benzyl alcohol.

It is contemplated that the staining composition can be produced bycombining sterile agents to one another so that the resulting stainingcomposition is sterile. However, in another approach, once the tissuestaining composition has been prepared, it can be sterilized, forexample, by filter sterilization, by exposure to one or more sterilizingagents, separately or in combination, such as exposure to hightemperature and/or high pressure (for example, during autoclaving), orby exposure to ionizing radiation. Alternatively, the tissue stainingcomposition after inclusion in a delivery device (e.g., a vial, syringe,etc.) can be sterilized using one or more procedures known and used inthe art of sterilization, for example, by exposure to one or moresterilizing agents, separately or in combination, such as exposure tohigh temperature and/or high pressure (for example, during autoclaving),or by exposure to ionizing radiation. The sterilization processpreferably achieves a sterility assurance level (SAL) of 10⁻³ or better;i.e. the probability of any given unit of product being non sterileafter the process is no more than 1 in 10³. More preferably, thesterilization process achieves an SAL of 10⁻⁴ or better, 10⁻⁵ or better,10⁻⁶ or better, or 10⁻⁷ or better.

In another aspect, the invention provides a method of preparing theforegoing tissue staining composition, the method comprising: (a)producing a composition comprising carbon particles, preferablydepyrogenated carbon particles, having a mean particle diameter of lessthan 0.3 μm or less than 0.2 μm in diameter; and (b) combining thecomposition comprising the carbon particles with an optional ASA, and anoptional mucoadhesive agent and/or one or more other excipients toproduce the tissue staining composition. During step (a), the carbonparticles preferably are combined with a surfactant and an anti-foamingagent prior to a deagglomeration procedure that produces carbonparticles to give a mean particle diameter of less than 0.3 μm or lessthan 0.2 For example, the carbon particles produced in step (a) can beproduced by sonicating or homogenizing carbon particles (which includecarbon primary particles, aggregates, and agglomerates) having a meanparticle diameter of, for example, greater than 5.0 μm in diameter toproduce carbon particles having a mean particle diameter of less than0.3 μm or less than 0.2 μm in diameter.

The resulting composition can be sterilized, for example, by filtersterilization, autoclaving or exposure to ionizing radiation.Furthermore, when the composition is introduced into a delivery device,the resulting device can be terminally sterilized by using one or moreof the sterilization approaches described herein.

In another aspect, the invention provides a method of staining a regionof tissue in a subject, the method comprising injecting the foregoingtissue staining composition into the region of interest in the subjectin an amount effective to stain the region so as to be visible by visualinspection, for example, by the unaided eye, an endoscope, laparoscopeor other device.

In one embodiment, the tissue is present in the gastrointestinal tract,bladder, breast, lymph nodes, lung, or central or peripheral nervoussystem of the subject. In certain embodiments, the tissue is present inthe gastrointestinal tract of the subject.

In another embodiment, the stain is visible, for example, visibleendoscopically, for at least 6 months, 12 months, 24 months, 36 months,48 months, 60 months, or 120 months after the staining of tissue.

These and other aspects and features of the invention are described inthe following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages of theinvention will become apparent from the following description ofpreferred embodiments, as illustrated in the accompanying drawings. Likereferenced elements identify common features in the correspondingdrawings. The drawings are not necessarily to scale, with emphasisinstead being placed on illustrating the principles of the presentinvention, in which:

FIG. 1 illustrates the development and features of exemplary stainingsolutions of the invention.

FIG. 2 is a bar chart showing the effect of carbon particle size andconcentration on the darkness of tissue staining solution.

FIG. 3 is a bar chart showing that the anti-settling properties of acarbon particle-based tissue staining solution containing 0.50% (w/v)carbon particles is based upon the combination of particle size and thepresence of a sufficient amount of an ASA.

FIG. 4 is a bar chart showing that the anti-settling properties of acarbon particle-based tissue staining solution containing 0.25% (w/v)carbon particles is based upon the combination of particle size and thepresence of a sufficient amount of an ASA.

FIG. 5 is a bar chart showing the effect of carbon particle size andconcentration on the darkness of tissue staining solutions containingthe ASA, HEC 2K.

FIG. 6 is a bar chart showing the effect of carbon particle size andconcentration on the darkness of tissue staining solutions containingthe ASA, HEC 5K.

FIG. 7 is a bar chart showing the mucoadhesive properties of exemplarycarbon particle-based tissue staining solutions based upon migrationspeed along a tilted mucin/agar gel.

FIGS. 8A and 8B are pictures visually showing the darkness andmucoadhesive properties of various staining solutions containing eitherHEC 5K before the agar/mucin plate was tilted (FIG. 8A) or after theplate was tilted (FIG. 8B).

DETAILED DESCRIPTION

The invention is based at least, in part, upon the discovery that it ispossible to produce a biocompatible, carbon particle-based tissue stainthat is reliable to use, easily observable during injection into tissue,and provides a durable, reliable mark that can be visualized over time,either by direct observation or by endoscopic or laparoscopicinspection. This has been facilitated by the discovery that, when usingcarbon particles as the staining agent, it is important to use carbonparticles that have a mean particle diameter less than 0.3 μm (forexample, less than 0.2 μm in diameter) to enhance the darkness of thestaining agent.

Depending upon the presence of an agent that reduces or eliminatescarbon particle agglomeration (for example a surfactant, such as anon-ionic surfactant), the carbon particle may remain in suspension forprolonged periods of time with little or no sedimentation (settling).However, depending upon the circumstances, the composition may alsocomprise an anti-settling agent and/or a mucoadhesive agent. A singleagent can have both anti-settling and mucoadhesive properties. Theresulting tissue stain is much darker than comparable carbonparticle-based tissue stains that contain a larger amount of carbonparticles where the carbon particles have a larger mean particlediameter. As a result, the tissue stains of the invention are easier tovisualize at the time of injection into a region of tissue of interest,and provide a durable mark at the region that can be visualized overtime. Furthermore, the resulting tissue stain has improved handling andstorage characteristics because the carbon particles are less likely tosettle out of solution upon storage and can be easily in injected intotissues of interest using commercially available needles and injectionsystems.

The terms “tissue stain,” “tissue staining composition,” and “tissuemarking composition” are used interchangeably herein.

The properties and advantages of the carbon partible-based tissuestaining compositions of the invention are shown schematically inFIG. 1. In particular, the tissue staining compositions of the invention(i) are much darker (achieved by reducing the size of the carbonparticles) and thus are easier to visualize upon introduction into thetissue of interest and then produce a durable mark that can bevisualized over time, (ii) exhibit reduced settling of the carbonparticles (achieved by reducing the particle size and optionally byadding an anti-settling agent) and thus are easier to manipulate by theuser, and (iii) are mucoadhesive (achieved by adding a mucoadhesiveagent and/or by using an anti-settling agent with mucoadhesiveproperties) and thus are contemplated to be easier to visualize uponintroduction into a tissue of interest with less “bleeding” of the stainout of the site of introduction and produce a durable mark that can bevisualized over time.

The tissue stains of the invention can be easily visualized as the stainis being injected into a region of tissue of interest, and whenintroduced, given the darkness and the optional mucoadhesive propertiesof the tissue stain, the stain can be visualized, for example, by directobservation or by a medical instrument, for example, endoscope orlaparoscope, over a prolonged period of time, for example, for weeks,months or years. The tissue stains are particularly useful when aphysician or other medical practitioner wishes to mark a tissue forsubsequent surgery or to monitor a region of a tissue over time, forexample, to assess whether potentially abnormal tissue has becomepre-cancerous or cancerous.

For example, if a cancerous or precancerous lesion is found in thegastrointestinal tract, urinary bladder, bronchi of the lungs, breasttissue, or lymph nodes or in other locations, a marking at the site canbe used to guide a surgeon to that site in a subsequent procedure. Byway of example, polyps in the colon typically are removed promptlybecause of their potential for malignancy. Polyps are discrete masslesions that protrude into the intestinal lumen. Mucosal neoplastic(adenomatous) polyps give rise to adenocarcinoma of the colon andtherefore, polyps detected at sigmoidoscopy or barium enema are removedas soon as possible by colonoscopic polypectomy or other techniques suchas those described in U.S. Pat. Nos. 5,122,147 and 5,542,948. Sometimespolyps or tumors cannot be safely or completely removed by colonoscopy,and surgical resection must follow. Once the polyps have been removed,surveillance colonoscopy is periodically repeated to look for missedpolyps, new adenomas, and residual or recurrent cancer.

It is understood that the tissue staining compositions can be used tomark a variety of tissues in a subject, for example, tissues in thegastrointestinal tract, the breast, the lymph nodes, the bladder andurinary tract, etc. However, as discussed in more detail below, aparticular tissue staining formulation can be optimized for delivery toand/or staining a tissue of interest.

I. Carbon Particles

The tissue marking composition of the invention includes carbonparticles as a source of pigment. Although the intensity of the color(darkness) can be increased by increasing the concentration of carbonparticles in suspension, it has also been discovered that reducing theparticle size can have an even more profound effect on the darkness ofthe suspension. As a result, by reducing particle size, for example, bydeagglomeration, it is possible to produce staining compositions thatare much darker than comparable staining compositions with much higherconcentrations of particles that are larger in size. This feature isdemonstrated in FIGS. 2, 5, 6 and 8. The reduction in the particle sizemay also reduce the rate of settling of the particles in solution.Furthermore, depending upon the size of the particles example, particleshaving a mean particle diameter less than 0.3 μm, or preferably lessthan 0.2 μm) and the presence of an agent, for example, a surfactant(for example, a non-ionic surfactant) that reduces or preventsreagglomeration of the carbon particles, it may be possible to produce astain where the carbon particles remain in suspension for a prolongedperiod of time. Although filtering of particles can reduce the size ofthe particles included in the formulation, it does not actually changethe size of the agglomerates and the minimum size of the particles islimited by the size of the filter.

As used herein, the term “carbon particles” is understood to meanindividual carbon particles (primary carbon particles) as well asaggregates of individual carbon particles where the aggregates ofsmaller particles are bound together strongly enough to behave as asingle particle, both of which have a particle size that cannot bereduced by exposure to mechanical forces and/or energy, for example, bysonication or homogenization, for example, by using the proceduresdescribed herein. Individual carbon particles and/or aggregates may beheld together by weak bonds (for example, van der Waals forces) to formagglomerates that can be broken by the addition of mechanical forcesand/or energy (for example, via sonication or homogenization) to producepredominantly individual carbon particles, aggregates and combinationsthereof (e.g., agglomerates having a mean diameter greater than 5 μmrepresent less than 30%, 20%, 10%, or 3%, 2% or 1% of the composition).

In certain embodiments, the carbon particles, which optionally have beenexposed to a deagglomeration process, have a mean particle diameter lessthan 0.3 μm in diameter, for example, less than 0.2 μm in diameter. Inthe tissue staining compositions of the invention, the carbon particles(which can be primary carbon particles, aggregates of carbon particles,or a combination thereof) have a mean particle diameter less than 0.3 μmin diameter, preferably less than 0.2 μm in diameter. The size of thecarbon particles can be measured using techniques known in the art suchas by dynamic light scattering, laser light scattering particle sizeanalysis or by disc centrifuge analysis. The carbon particles may have amean particle diameter in the range from about 0.1 μm to less than 0.3μm. In certain embodiments, the carbon particles have a mean particlediameter in the range from about 0.1 μm to about 0.2 μm. In certainembodiments, the carbon particles have a mean particle diameter in therange from about 0.15 μm to about 0.2 μm. In certain embodiments, themean particle diameter is less than 0.2 μm.

In certain embodiments, the carbon particles have a mean diameter in therange from about 0.01 μm to less than 0.3 μm, from about 0.01 μm to lessthan 0.25 μm, from about 0.01 μm to less than 0.2 μm, from 0.01 μm toless than 0.15 μm.

In certain embodiments, the carbon particles have a mean diameter fromabout 0.05 μm to 0.25 μm or less, from about 0.05 μm to 0.2 μm or less(for example, from 0.05 μm to 0.19 μm, from 0.05 μm to 0.18 μm, from0.05 μm to 0.17 μm, from 0.05 μm to 0.16 μm, from 0.05 μm to 0.15 μm,from 0.05 μm to 0.14 μm, from 0.05 pin to 0.13 pin, from 0.05 μm to 0.12μm, from 0.05 μm to 0.11 μm, or from 0.05 μm to 0.1 μm). In certainembodiments, the carbon particles have a mean diameter from about 0.08μm to 0.2 μm or less (for example, from 0.08 μm to 0.19 μm, from 0.08 μmto 0.18 μm, from 0.08 μm to 0.17 μm, from 0.08 μm to 0.16 μm, from 0.08μm to 0.15 μm, from 0.08 μm to 0.14 μm, from 0.08 μm to 0.13 μm, from0.08 μm to 0.12 μm, from 0.08 μm to 0.11 μm, or from 0.08 μm to 0.1 μm).In certain embodiments, the carbon particles have a mean diameter from0.1 μm to 0.2 μm or less (for example, from 0.1 μm to 0.19 μm, from 0.1μm to 0.1.8 μm, from 0.1 μm to 0.17 μm, from 0.1 μm to 0.16 μm, from0.1. μm to 0.15 μm, from 0.1 μm to 0.14 μm, from 0.1 μm to 0.13 μm, orfrom 0.1 μm to 0.12 μm, or from 0.1 μm to 0.11 μm).

Carbon particles can be derived from carbon black, charcoal, or coke.Carbon black is finely divided carbon such as vaporized heavy oilfractions produced by burning hydrocarbons using partial oxidation. Thepigment can contain over 97% carbon. The oil furnace process representsthe most widely used method for producing carbon black. Generally, aliquid hydrocarbonaceous feedstock is sprayed into turbulent products ofcombustion produced by reacting fluid fuel and oxygen and thehydrocarbon feedstock is converted into carbon black which is separatedfrom combustion gases. Carbon black can also be produced by burningnatural gas and letting the flame impinge on a cool surface. Thepreferred carbon black useful herein is low in incompletely burnedhydrocarbons which may be absorbed during manufacture; particularly, thecarbon black is low in aromatics and other compounds which may becarcinogens. More particularly, the preferred carbon black is low inresidual polycyclic aromatic hydrocarbons. By “low” is meantsubstantially non-carcinogenic levels. In a preferred embodiment, thecarbon black has a level of polycyclic aromatic hydrocarbons of notgreater than 0.5 ppm based on the amount of carbon black.

Charcoal can be prepared by the ignition of wood, sugar, and othercarbon-containing compounds in the absence of air. It has a graphiticstructure but is not well developed in crystallinity. It will thereforebe categorized as amorphous herein. Activated carbon is similar obtainedby the carbonization or destructive distillation of vegetable matter,e.g., wood, nut shells, bones, or other carbonaceous material. Thecarbon is activated by heating to high temperatures in the presence ofwater or carbon dioxide which results in a carbon having a porousinternal structure. Carbon which has not been subjected to thistreatment will be called unactivated herein. Coke is prepared by heatingcoal in the absence of air.

Exemplary carbon particles, preferably 4750 Monarch, can be obtainedfrom Cabot Corp., Billerica, Mass. or Asbury Carbon 5388 or 5377obtained from Asbury Carbons, Asbury, N.J.

The density of the carbon particles typically used in the tissuestaining composition of the invention is from about 1.7 g/cm³ to about1.9 g/cm³, which is greater than the density of water either alone orwith glycerol. As a representative formulation vehicle, the density ofwater containing 25% (w/v) glycerol is not appreciably greater than 1.0g/cm³. Due to the higher density of the carbon particles than the liquidphase, the carbon particles typically settle out to some degree,resulting in a liquid portion and a settled solid portion, such that theliquid portion has a lower number of carbon particles compared with theoriginal composition as a whole. A reduced amount of carbon particlescan lead to poorer marking performance of the liquid portion of thecomposition. Such solutions should be shaken, vortexed or otherwisemixed prior to use. Given that some users may not redistribute thestaining agent (for example, by shaking) prior to use, it is desirableto reduce the amount of settling of the carbon particles over time.Methods for reducing settling include reducing the particle size, eitheralone or in combination with increasing the liquid viscosity of thecomposition and/or including an anti-settling agent. It has beendiscovered that the rate of settling of the carbon particles can beslowed significantly by reducing the mean particle diameter (includingaggregates) to less than 0.3 μm in diameter (for example, less than 0.2μm in diameter) and optionally including an anti-settling agent (see,FIGS. 3 and 4).

Typically carbon is available as primary carbon particles, aggregates,agglomerates, and combinations thereof. The primary carbon particles andthe aggregates cannot be reduced in size, for example, by theapplication of mechanical forces and/or energy, for example, viasonication or homogenization. Given that the carbon often comprisesagglomerates (for example, agglomerates having a mean particle diametergreater than 5 μm) the agglomerates can be broken down by adeagglomeration process to liberate individual carbon particles and/oraggregates. When the attractive forces that hold the agglomeratestogether are disrupted, for example, mechanically and/or throughapplication of energy, the resulting carbon particles can have a meanparticle diameter less than 0.3 μm, for example, less than 0.2 μm, orhave a mean particle diameter in a range of from about 0.1 to about 0.2μm, or have a mean diameter in a range of from about 0.05 μm to lessthan 0.2 μm. The smaller particles appear much darker than a comparableweight of larger particles. Without wishing to be bound by theory, it isbelieved that the visibility of a given weight of carbon particlesdiminishes as the particles became larger, because the eye observesfewer large particles as being less black than the same weight of morenumerous smaller carbon particles, due to the larger “spaces” betweenthe larger particles. Thus, deagglomeration can increase the visibilityof the mark, as well as reduce the amount and speed of settling. Thesize of the particles can be determined by using a number of approachesincluding, for example, laser light scattering particle analysis (LLSPA)(also known as laser diffraction or laser diffractometry), and dynamiclight scattering (DLS). LLSPA may be more accurate when analyzing largerparticles (for example, particles having a mean diameter of about 5 μmor greater) whereas DLS may be more accurate when analyzing smallerparticles (for example, particles having a mean diameter of 0.5 μm orless). In addition, particle sizes can be measured using a LUMiSizer 650that employs Space- and Time-resolved Extinction Profiles (STEP)technology, which measures particles in the range of, for example, 20 nmto 100 μm.

The darkness of the tissue staining compositions containing the carbonparticles can be measured as a function of absorbance or reflectance bymeans of a densitometer or uv/visible spectrophotometer, such as an XRite 504 Portable Color Reflection Spectrodensitometer and a ThermoScientific Biomate S3 UV-Vis spectrophotometer and the darkness may beexpressed in terms of % Absorbance, % Reflectance, or optical density.

The deagglomeration process can be achieved by sonication,homogenization, high shear mixing, or ball milling, or other approachesto reduce particle size. Preferably, deagglomeration is achieved bysonication or homogenization. In certain embodiments, the carbon isdeagglomerated by sonication in a 350 W Biologics Model 3000 UltrasonicHomogenizer with Solid Titanium Tip ½″ Diameter unit from BioLogics,Inc., Manassas, Va. sonicator on 60% power, for about 3 minutes or untilapproximate particle size is achieved. In other embodiments, the carbonis deagglomerated by homogenization, for example, by homogenizing atapproximately 8,500 rpm for about 1 hour (for example, using a SilversonModel L5M-A mixer-homogenizer) or until the approximate particle size isachieved. One exemplary approach for deaggregating carbon particles byhomogenization is described in Example 2.

In certain embodiments, the carbon particles are deagglomerated beforethey are mixed with other excipients, for example, the ASA and theoptional mucoadhesive agent, because the process of deagglomeration mayresult in viscosity changes to ASA and the mucoadhesive agent,especially if the ASA or mucoadhesive agent are fabricated from orotherwise contain polymers which can be altered by high shearconditions. The carbon particles may be maintained as aggregates havinga mean particle diameter less than 0.3 μm (for example, less than 0.2μm) by the inclusion of additional additives, for example, one or moresurfactants described below, which prevent or slow down the rate ofreagglomeration due to van der Waals attraction.

Preferably the carbon particles are depyrogenated or are otherwisepyrogen free. This can be achieved, for example, by heating carbonpowder at 220° C. for 1 hour to produce pyrogen free dry carbon.

Depending upon the intended application, the final concentration ofcarbon in the tissue staining composition can be from about 0.025% (w/v)to about 2.0% (w/v), from about 0.025% (w/v) to about 1.5% (w/v), fromabout 0.025% (w/v) to about 1.0% (w/v), from about 0.025% (w/v) to about0.75% (w/v), from about 0.025% (w/v) to about 0.5% (w/v), 0.1% (w/v) toabout 2.0% (w/v), from about 0.1% (w/v) to about 1.5% (w/v), from about0.1% (w/v) to about 1.0% (w/v), from about 0.1% (w/v) to about 0.75%(w/v), from about 0.1% (w/v) to about 0.5% (w/v), from about 0.25% (w/v)to about 2.0% (w/v), from about 0.25% (w/v) to about 1.5% (w/v), fromabout 0.25% (w/v) to about 1.0% (w/v), from about 0.25% (w/v) to about0.75% (w/v), from about 0.25% (w/v) to about 0.5% (w/v), from about 0.5%(w/v) to about 2.0% (w/v), from about 0.5% (w/v) to about 1.5% (w/v),from about 0.5% (w/v) to about 1.0% (w/v), from about 0.5% (w/v) toabout 0.75% (w/v), from about 0.75% (w/v) to about 2.0% (w/v), fromabout 0.75% (w/v) to about 1.5% (w/v), from about 0.75% (w/v) to about1.0% (w/v), from about 1.0% (w/v) to about 2.0% (w/v), or from about1.0% (w/v) to about 1.5% (w/v).

In certain preferred embodiments, the final concentration of carbon,preferably deagglomerated carbon present in the tissue stainingcomposition ranges from about 0.25% (w/v) to about 0.5% (w/v). However,when a darker mark is desired, the final concentration of carbon presentcan be increased to a range from about 0.5% (w/v) to about 1.0% (w/v).

II. Anti-Settling Agents (ASAs)

In addition to agents (for example, surfactants, such as non-ionicsurfactants) that reduce or prevent the reagglomeration of the carbonparticles, the tissue marking compositions optionally may also includean anti-settling agent (ASA) to aid in preventing or reducing thesettling of the carbon particles. Many ASAs are available, however theymay be incompatible with other ingredients in the composition and/or themethods of making or sterilizing the composition. Preferred ASAs arethermally stable, for example, to facilitate sterilization byautoclaving, and should reduce the settling speed of carbon particleshaving a mean particle diameter of less than 0.3 μm in diameter (forexample, less than 0.2 μm in diameter).

Given their lack of thermal stability in water-based solution underautoclaving conditions, hydroxypropylmethyl cellulose, non-crosslinkedhyaluronic acid, xanthan, pectin, tragacanth gum, andpolyvinylpyrrolidone should not be included in the composition if it isgoing to be sterilized by autoclaving. Suitable ASAs for use in thecomposition which can be sterilized by autoclaving include hydroxyethylcellulose, hydroxypropyl cellulose, dextran, and guar gum. Hydroxyethylcellulose, hydroxypropyl cellulose, dextran, and guar gum also havemucoadhesive properties (see, TABLES 3 and 4).

Exemplary hydroxyethyl cellulose (HEC), includes hydroxyethyl cellulosehaving a molecular weight of approximately 1.0×10⁶ Da with a viscosityof approximately 2000 cp at 1% (w/v) (HEC2K) and hydroxyethyl cellulosehaving a molecular weight of approximately 1.3×10⁶ Da with a viscosityof approximately 5000 cp at 1% (w/v) (HEC5K), which can be obtained fromSpectrum Chemical or Ashland. Exemplary hydroxypropyl cellulose (HPC),includes hydroxypropyl cellulose having a molecular weight ofapproximately 8.5×10⁵ Da with a viscosity of approximately 4950 cp at 5%(w/v), which can be obtained from Spectrum Chemical or Ashland.

The concentration of the ASA in the final product can range from about0.025% (w/v) to less than 5.0% (w/v), from about 0.025% (w/v) to about2.0% (w/v), from about 0.025% (w/v) to about 1.5% (w/v), from about0.025% (w/v) to about 1.0% (w/v), from about 0.025% (w/v) to about 0.75%(w/v), from about 0.025% (w/v) to about 0.5% (w/v), from about 0.05%(w/v) to about 1.5% (w/v), from about 0.05% (w/v) to about 1.0% (w/v),from about 0.05% (w/v) to about 0.75% (w/v), from about 0.05% (w/v) toabout 0.5% (w/v), from about 0.075% (w/v) to about 1.5% (w/v), fromabout 0.075% (w/v) to about 1.0% (w/v), from about 0.075% (w/v) to about0.75% (w/v), from about 0.075% (w/v) to about 0.5% (w/v), from about0.1% (w/v) to about 1.5% (w/v), from about 0.1% (w/v) to about 1.0%(w/v), from about 0.1% (w/v) to about 0.75% (w/v), from about 0.1% (w/v)to about 0.5% (w/v), from about 0.5% (w/v) to about 2.0% (w/v), fromabout 0.5% (w/v) to about 1.5% (w/v), from about 0.5% (w/v) to about1.0% (w/v), from about 0.5% (w/v) to about 0.75% (w/v), from about 0.75%(w/v) to about 2.0% (w/v), from about 0.75% (w/v) to about 1.5% (w/v),from about 0.75% (w/v) to about 1.0% (w/v), from about 1.0% (w/v) toabout 2.0% (w/v), or from about 1.0% (w/v) to about 1.5% (w/v).

As shown in Example 2, the combination of such ASAs with carbonparticles having mean particle diameter of less than about 0.3 μm canresult in much slower settling rates than other carbon particle-basedliquid endoscopic tissue staining compositions. Settling is a processthat takes place over time, and may proceed over a period of minutes,hours, days, months, or years. It is possible to quantify the rate ofsettling using a variety of approaches, for example, via centrifugationto accelerate the settling process. Similarly, the rate of settling, aswell as particle size analysis, can be determined using a LUMiSizer 650available from LUM GmbH, Berlin, Germany.

As a comparative method of analyzing the settling properties of acomposition, a composition of interest may be subjected to centrifugalforces many times the force of gravity which allows the simulation ofmonths of settling to occur in minutes to hours. Centrifugation of atube containing a sample of interest may result in settling of thecarbon particles at the bottom of the tube leaving a liquid depleted ofcarbon particles at the upper most regions of the liquid. A portion ofthe liquid in the upper most regions of the liquid (for example, theupper third portion) can then be analyzed to determine the amount ofcarbon particles that have settled. In certain embodiments, the stainingcomposition can be characterized in that, when subjected tocentrifugation for 60 minutes at 5,000×g, the staining composition,which includes the ASA, exhibits less than 70%, less than 60%, less than50%, less than 40%, less than 30%, or less than 20% of the settling whencompared against similar composition where the particles have a meanparticle diameter greater than 5 μm and/or the composition lacks an ASA.

III. Mucoadhesives

Depending upon the choice of the ASA (for example, an ASA that does notalso have mucoadhesive properties), it may be preferable to include anadditional mucoadhesive agent. The mucoadhesive agent can aid theadherence of the staining agent in the marking of the composition to thetarget tissue. Without wishing to be bound by theory, it is contemplatedthat greater mucoadhesion promotes cohesion of the mark and, therefore,increases the likelihood that the mark will remain in one place and bemore readily visible near the area of interest, potentially by reducingspreading of the pigment over time. Furthermore, it is also believedthat increased cohesion may reduce the leakage associated with markingsolutions post injection, such leakage being undesirable as it can cloudthe field of view of an endoscope and require an operating physician torinse the area being viewed.

Preferred mucoadhesive agents optionally are thermally stable,compatible with other components in the formulation and increase themucoadhesion of carbon particles with a mean particle diameter of lessthan 0.3 μm in diameter. Exemplary mucoadhesive agents are described inSaraswathi et al. (2013) INT. J. PHARM. PHARM. SCI. 5:423-430. Exemplarymucoadhesive agents can be selected from the group consisting ofhydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, collagen, gelatin, albumin, alginate, chitosan, dextran, guargum, polyethylene glycol (for example, polyethylene glycol200,000-400,000 daltons), polylactic acid, and polyglycolic acid.

The mucoadhesives can be present in the staining composition at a finalconcentration of from about 0.025% (w/v) to less than 5.0% (w/v), fromabout 0.025% (w/v) to about 2.0% (w/v), from about 0.025% (w/v) to about1.5% (w/v), from about 0.025% (w/v) to about 1.0% (w/v), from about0.025% (w/v) to about 0.75% (w/v), from about 0.025% (w/v) to about 0.5%(w/v), from about 0.05% (w/v) to about 1.5% (w/v), from about 0.05%(w/v) to about 1.0% (w/v), from about 0.05% (w/v) to about 0.75% (w/v),from about 0.05% (w/v) to about 0.5% (w/v), from about 0.075% (w/v) toabout 1.5% (w/v), from about 0.075% (w/v) to about 1.0% (w/v), fromabout 0.075% (w/v) to about 0.75% (w/v), from about 0.075% (w/v) toabout 0.5% (w/v), from about 0.1% (w/v) to about 1.5% (w/v), from about0.1% (w/v) to about 1.0% (w/v), from about 0.1% (w/v) to about 0.75%(w/v), from about 0.1% (w/v) to about 0.5% (w/v), from about 0.5% (w/v)to about 2.0% (w/v), from about 0.5% (w/v) to about 1.5% (w/v), fromabout 0.5% (w/v) to about 1.0% (w/v), from about 0.5% (w/v) to about0.75% (w/v), from about 0.75% (w/v) to about 2.0% (w/v), from about0.75% (w/v) to about 1.5% (w/v), from about 0.75% (w/v) to about 1.0%(w/v), from about 1.0% (w/v) to about 2.0% (w/v), or from about 1.0%(w/v) to about 1.5% (w/v).

In certain embodiments, when tested for mucoadhesive properties, forexample as described in Example 3, the staining composition including amucoadhesive agent (either by means of the ASA or a separatemucoadhesive agent) shows a reduction of travel speed along a tilted(for example, tilted at 30° from horizontal) surface containing 2% mucinand 2% agar (simulates gastric tissue lining) by at least 60%, at least50%, at least 40%, at least 30%, or at least 20% compared to a similarcomposition lacking the mucoadhesive agent.

IV. Viscosity-Increasing Agents

The composition optionally may also include a viscosity-increasing agentto prevent or otherwise reduce the settling of the carbon particles, andto adjust the viscosity of the composition to a desired level. Aviscosity greater than that of water may slow down the settling processbut viscosities too high may result in a solution that cannot beinjected through a suitable delivery needle, for example, a 25 gaugeneedle. In certain embodiments, the staining composition is capable ofpassing through a 25 gauge needle 240 cm in length upon application of aforce no greater than about 32N, optionally from about 22 to about 32N,or about 22N or less, or about 10N or less, to push the compositionthrough the needle.

Preferred viscosity-increasing agents include, for example, glycerol,propylene glycol, isopropylene glycol, polyethylene glycol, cellulose,carboxymethyl cellulose, hydroxypropyl methylcellulose, and hyaluronicacid. In certain embodiments the agent is thermally stable andcompatible with other formulation components to prevent the carbonparticles from rapidly (e.g., less than 60 minutes, 30 minutes, 20minutes, 10 minutes, or 5 minutes) settling out of solution. In certainembodiments, the viscosity-increasing agent is glycerol. To the extentthat the viscosity-increasing agents are not thermally stable, forexample, carboxymethyl cellulose and hydroxypropyl methylcellulose, thetissue staining solution may be filter sterilized or other non-thermalapproach.

The viscosity-increasing agents can be present in the stainingcomposition at a final concentration of from about 5% to about 25%, 5%to about 20%, 5% to about 15%, 5% to about 10%, 10% to about 25%, 10% toabout 20%, 10% to about 15%, 15% to about 25%, or 15% to about 20%(w/v).

V. Additional Additives

The composition may also include other additives, for example, asurfactant, an anti-foaming agent, and/or a preservative.

The compositions optionally comprise a surfactant to reduce or eliminatethe reagglomeration of the carbon particles and/or to wet the carbonparticles in water-based solvents. The surfactant allows the carbonagglomerates to be more readily dispersed in aqueous solutions andprohibits reagglomeration after sonication/homogenization. In certainembodiments the surfactant is a non-ionic surfactant. In certainembodiments, the surfactant is selected from the group consisting ofpolyethoxylated sorbitan ester and/or sorbitan ester. Exemplarypolyethoxylated sorbitan esters include, for example, PEG-20 sorbitanmonolaurate (Tween® 20), PEG-4 sorbitan monolaurate (Tween® 21), PEG-20sorbitan monopalmitate (Tween® 40), PEG-20 sorbitan monostearate (Tween®60), PEG-20 sorbitan tristearate (Tween® 65), and PEG-20 sorbitanmonooleate (Tween® 80), which are available commercially from CrodaEurope Ltd, England. In certain embodiments, PEG-20 sorbitan monooleate(Tween® 80) is preferred. Exemplary sorbitan esters include, forexample, Sorbitan monolaurate (Span® 20), Sorbitan monopalmitate (Span®40), Sorbitan monostearate (Span® 60), Sorbitan monooleate (Span® 80),Sorbitan sesquioleate (Span® 83), Sorbitan trioleate (Span® 85), andSorbitan isostearate (Span® 120).

In certain embodiments, the surfactant can be present at a finalconcentration of from about 0.01% to about 4.0% (w/v), from about 0.05%to about 2.0% (w/v), from about 0.05% to about 1.5% (w/v), from about0.05% to about 1.0% (w/v), from about 0.05% to about 0.75% (w/v), fromabout 0.5% to about 4% (w/v), from about 0.5% to about 2.0% (w/v), fromabout 0.5% to about 1.5% (w/v), from about 0.5% to about 1.0% (w/v),from about 0.5% to about 0.75% (w/v), from about 0.75% to about 4%(w/v), from about 0.75% to about 2% (w/v), from about 0.75% to about1.5% (w/v), or from about 0.75% to about 1.0% (w/v).

Preferred anti-foaming agents include, for example, a dimethicone andsimethicone. Simethicone (USP) comprises a mixture ofpoly(dimethylsiloxane) and silicon dioxide. The poly (dimethylsiloxane)is α-(trimethylsilyl)-ω-methyl-poly[oxy (dimethylsilylene)]. Theanti-foaming agent can be present at a final concentration from about0.01% to about 0.5% (w/v), from about 0.05% to about 0.25% (w/v), orfrog about 0.1% to about 0.2% (w/v).

The composition can also include a suitable preservative such as benzylalcohol, methyl or ethyl paraben, or benzalkonium chloride, which canfunction as an anti-microbial. In certain embodiments, the preservativeis present at a final concentration from about 0.01% to about 4.0%(w/v), from about 0.05% to about 2.0% (w/v), from about 0.05% to about1.5% (w/v), from about 0.05% to about 1.0% (w/v), from about 0.05% toabout 0.75% (w/v), from about 0.5% to about 4% (w/v), from about 0.5% toabout 2.0% (w/v), from about 0.5% to about 1.5% (w/v), from about 0.5%to about 1.0% (w/v), or from about 0.5% to about 0.75% (w/v).

Other pharmaceutically acceptable excipients may be added, e.g., bufferssuch as citrate or phosphate buffering agents. In certain embodiments,the composition does not contain significant amounts of shellac, phenol,polycyclic aromatic hydrocarbons, ammonia, or gelatins.

The pH of the tissue staining composition should be compatible withstaining living tissue, and preferably has a pH in the range from about6 to about 8.

It is understood that each of the foregoing components of the tissuestaining solution including, for example, the carbon particles, ASAs,mucoadhesives, viscosity increasing agents, and additional additives arepharmaceutically acceptable. The term “pharmaceutically acceptable” asused herein refers to those compounds, materials, compositions, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

VI. Methods of Manufacture and Use

The tissue staining compositions can be prepared using a variety ofapproaches.

In a first approach, the marking compositions are produced by making asolution comprising carbon particles having a mean particle diameterless than 0.3 μm, preferably less than 0.2 μm. This can be achieved by,for example, sonicating or homogenizing a carbon particle containingsolution, as discussed above. Thereafter, the particles are combinedwith other excipients, for example, an ASA, a mucoadhesive agent, asurfactant, and other agents etc.

In an exemplary protocol for making a tissue stain containing an ASA, afirst pre-mix solution (the carbon pre-mix) is prepared by dissolving asurfactant (e.g., Tween® 80) and an anti-foaming agent (e.g.,simethicone) in an aqueous solution (e.g., water), then addingdepyrogenated carbon particles and mixing the surfactant, anti-foamingagent and carbon particles to produce a surfactant/carbon particlemixture. The surfactant/carbon particle mixture is then exposed to adeagglomeration procedure (for example, by sonication and/orhomogenization) to produce the first pre-mix solution. In this method,the surfactant coats the carbon particles to slow down or preventagglomeration or reagglomeration of the carbon particles. A secondpre-mix solution (the polymer pre-mix) is prepared by combining the ASA(e.g., HEC) with the optional viscosity-increasing agent (e.g.,glycerol) by mixing. The glycerol can also wet the ASA (e.g., HEC). Theresulting mixture (or the ASA alone) is combined with an aqueoussolution (e.g., water). Preferably the ASA (e.g., HEC) is not exposed tothe deagglomeration procedure as this may shear the polymer therebyreducing or otherwise eliminating the beneficial properties of thepolymer. In other words, the carbon particles are deagglomerated beforebeing mixed with the ASA. A preservative can be added to the firstpre-mix solution, the second pre-mix solution or both the first andsecond pre-mix solutions. The first and second pre-mix solutions thenare combined, mixed, and introduced into a suitable container ordelivery device, for example, a syringe.

If the final tissue stain lacks the optional ASA and/orviscosity-increasing agent, then the second pre-mix solution can lackthe ASA and/or viscosity-increasing agent. An example of a protocol formaking a tissue stain without an ASA is described in Example 2. Briefly,a first pre-mix is prepared by combining a surfactant (e.g., Tween® 80),an anti-foaming agent (e.g., simethicone) and a viscosity increasingagent (e.g., glycerol) in water, and then adding the resulting mixtureto carbon particles with mixing to produce the first pre-mix. Apreservative can be added to the first pre-mix. A second pre-mix isprepared by adding a surfactant (e.g., Tween® 80) to water withstirring. Then a viscosity increasing agent (e.g., glycerol) is added tothe mixture to produce the second pre-mix. A preservative can be addedto the second pre-mix. Thereafter, the first pre-mix and the secondpre-mix are combined with mixing (homogenization) at the appropriaterate and for the appropriate time, for example, for over 20 hours, untila tissue stain containing carbon particles with the desired particlesizes are obtained.

In a second approach, the deagglomerated carbon particles are mixed withthe excipients, for example, the optional ASA, mucoadhesive agent,surfactant, etc. and then the carbon particles are filtered with a 0.2μm filter (or a filter with smaller pore sizes) and size sorted to havea mean particle diameter less than 0.3 μm or less than 0.2 μm.

The resulting tissue staining compositions created by the foregoingapproaches and protocols, when introduced in a suitable container, canthen be sterilized to maximize shelf life using sterilization techniquesknown in the art, including, as appropriate, autoclaving, exposure toionizing radiation or by sterile filtration. Similarly, once the tissuestaining composition has been included in a delivery device, thedelivery device may be terminally sterilized using one or moreprocedures known in the art of terminal sterilization, for example, byautoclaving or by exposure to ionizing radiation.

The sterilization process preferably achieves a sterility assurancelevel (SAL) of 10⁻³ or better; i.e. the probability of any given unit ofproduct being non sterile after the process is no more than 1 in 10³.More preferably, the sterilization process achieves an SAL of 10⁻⁴ orbetter, 10⁻⁵ or better, 10⁻⁶ or better, or 10⁻⁷ or better.

The staining composition can be used in the form of a liquid surgicalmarker for endoscopic and/or laparoscopic marking using known endoscopicand/or laparoscopic techniques. For example, the liquid tissue markingsolution can be drawn into a syringe and injected through a needle, forexample, a 25 gauge needle into the tissue of interest. In certainembodiments, the composition is capable of passing through a 25 gaugeneedle 240 cm in length by application of a force no greater than about32N, for example, from about 22N to about 32N, or about 22N or less, orabout 10N or less, or about 7N or less, or about 5N or less, to push thecomposition through the needle. The choice of the appropriate needleand/or injection system for introducing the tissue stain into the tissueof interest would be apparent to the user.

In certain embodiments, the tissue staining solutions do not includeemulsifiers and/or do not contain carbon particles encapsulated in anemulsifier and/or do not contain carbon particles encapsulated inemulsifier containing micelles, as described in U.S. Pat. No. 9,024,087.

An exemplary water based-tissue staining composition suitable formarking gastrointestinal tissue comprises:

-   -   (i) from about 0.025% to about 2.0% (w/v) carbon particles        having a mean particle diameter less than 0.3 μm or less than        0.2 μm; and    -   (ii) optionally from about 0.025% to less than 5.0% (w ASA        selected from hydroxyethyl cellulose (HEC), hydroxypropyl        cellulose (HPC), dextran, and guar gum, or a combination thereof        (for example, HEC2K or HEC5K or a combination thereof);    -   (iii) optionally from about 5.0% to about 25% (w/v) of a        viscosity-increasing agent (for example, glycerol);    -   (iv) optionally from about 0.1% to about 2.0% (w/v) of a        surfactant (for example, Tween® 80); and    -   (v) optionally from about 0.005% to about 1.0% (w/v) of an        anti-foaming agent (for example, simethicone).

An exemplary water based-tissue staining composition suitable formarking gastrointestinal tissue comprises:

-   -   (i) from about 0.025% to about 2.0% (w/v) carbon particles        having a mean particle diameter less than 0.3 μm or less than        0.2 μm; and    -   (ii) optionally from about 0.025% to less than 5.0% (w/v) of an        ASA selected from hydroxyethyl cellulose (HEC), hydroxypropyl        cellulose (HPC), dextran, and guar gum, or a combination thereof        (for example, HEC2K or HEC5K or a combination thereof);    -   (iii) from about 5.0% to about 2.0% (w/v) of a        viscosity-increasing agent (for example, glycerol);    -   (iv) from about 0.1% to about 2.0% (w/v) of a surfactant (for        example, Tween® 80); and    -   (v) from about 0.005% to about 1.0% (w/v) of an anti-foaming        agent (for example, simethicone).

Another exemplary water-based tissue staining composition suitable formarking gastrointestinal tissue comprises:

-   -   (i) 0.025% to 0.5% (w/v) carbon particles having a mean particle        diameter less than 0.3 μm or less than 0.2 μm;    -   (ii) optionally 0.1% to 0.5% (w/v) C, for example, HEC2K or        HEC5K or a combination thereof;    -   (iii) optionally about 15% glycerol;    -   (iv) optionally about 1% Tween® 80;    -   (v) optionally about 0.01% simethicone; and    -   (vi) optionally about 1% benzyl alcohol.

Another exemplary water-based tissue staining composition suitable formarking gastrointestinal tissue comprises:

-   -   (i) 0.025% to 0.5% (w/v) carbon particles having a mean particle        diameter less than 0.3 μm or less than 0.2 μm;    -   (ii) optionally 0.1% to 0.5% (w/v) C, for example, HEC2K or        HEC5K or a combination thereof;    -   (iii) about 15% glycerol;    -   (iv) about 1% Tween® 80;    -   (v) about 0.01% simethicone; and    -   (vi) about 1% benzyl alcohol.

Throughout the description, where apparatus, devices, and systems aredescribed as having, including, or comprising specific components, orwhere processes and methods are described as having, including, orcomprising specific steps, it is contemplated that, additionally, thereare apparatus, devices, and systems of the present invention thatconsist essentially of, or consist of, the recited components, and thatthere are processes and methods according to the present invention thatconsist essentially of, or consist of, the recited processing steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components.

Further, it should be understood that elements and/or features of acomposition or a method described herein can be combined in a variety ofways without departing from the spirit and scope of the presentinvention, whether explicit or implicit herein. For example, wherereference is made to a particular staining solution, that stainingsolution can be used in various embodiments of compositions of thepresent invention and/or in methods of the present invention, unlessotherwise understood from the context. In other words, within thisapplication, embodiments have been described and depicted in a way thatenables a clear and concise application to be written and drawn, but itis intended and will be appreciated that embodiments may be variouslycombined or separated without parting from the present teachings andinvention(s). For example, it will be appreciated that all featuresdescribed and depicted herein can be applicable to all aspects of theinvention(s) described and depicted herein.

It should be understood that the expression “at least one of” includesindividually each of the recited objects after the expression and thevarious combinations of two or more of the recited objects unlessotherwise understood from the context and use. The expression “and/or”in connection with three or more recited objects should be understood tohave the same meaning unless otherwise understood from the context.

The use of the term “include,” “includes,” “including,” “have,” “has,”“having,” “contain,” “contains,” or “containing,” including grammaticalequivalents thereof, should be understood generally as open-ended andnon-limiting, for example, not excluding additional unrecited elementsor steps, unless otherwise specifically stated or understood from thecontext.

Where the use of the term “about” is before a quantitative value, thepresent invention also includes the specific quantitative value itself,unless specifically stated otherwise. As used herein, the term “about”refers to a ±10% variation from the nominal value unless otherwiseindicated or inferred.

Where a molecular weight is provided and not an absolute value, forexample, of a polymer, then the molecular weight should be understood tobe an average molecule weight, unless otherwise stated or understoodfrom the context.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present invention remainsoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

At various places in the present specification, staining solutions,components, or features thereof are disclosed in groups or in ranges. Itis specifically intended that the description include each and everyindividual subcombination of the members of such groups and ranges. Byway of other examples, an integer in the range of 1 to 20 isspecifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

The use of any and all examples, or exemplary language herein, forexample, “such as” or “including,” is intended merely to illustratebetter the present invention and does not pose a limitation on the scopeof the invention unless claimed. No language in the specification shouldbe construed as indicating any non-claimed element as essential to thepractice of the present invention.

As a general matter, compositions specifying a percentage are by weightunless otherwise specified.

Practice of the invention will be more fully understood from theforegoing examples, which are presented herein for illustrative purposesonly, and should not be construed as limiting the invention in any way.

EXAMPLES Example 1—Effect of Carbon Particle Size on Darkness of TissueStain

This Example shows that carbon particle size has a profound impact onthe darkness of a tissue staining composition.

Various test solutions were prepared containing different concentrationsof carbon particles ranging from 0.025%-1.0% (w/v) of carbon particles(Monarch 4750 from Cabot Corp., Billerica Mass.) that were either notdeagglomerated and thus contained carbon particles having a meanparticle diameter of about 6.6 μm or deagglomerated by sonication toproduce carbon particles having a mean diameter less than 0.2 μm. Thebalance of each test solution included 15% (w/v) glycerol, 1% (w/v)Tween® 80, 1% (w/v) benzyl alcohol, 0.01% (w/v) simethicone, and sterilewater for injection (SWFI).

The test solutions were prepared as follows.

A first (2×) pre-mix was prepared by adding the following ingredients toSWFI at 80-100° C., one at a time, and mixing for 15 minutes beforeadding the next ingredient, to give the following final concentrationsof each ingredient in the pre-mix and in the order of addition—15% (w/v)glycerol, then 2% (w/v) Tween® 80, then 2% benzyl alcohol, then 0.02%(w/v) simethicone, then twice the required concentration of the carbonparticles desired in the final product when the first pre-mix was mixedwith an equal amount of a second pre-mix. The solution was mixed andallowed to cool to room temperature. For the non-sonicated samples, aportion of the resulting solution was stored to be combined with thesecond pre-mix. For the sonicated samples, a portion of the resultingsolution was sonicated using a 350 W Biologics Model 3000 UltrasonicHomogenizer with a solid ½″ diameter titanium tip (BioLogics, Inc.,Manassas Va.) for 3 minutes on 60% power prior to mixing with the secondpre-mix.

A second pre-mix was prepared by adding glycerol to SWFI at 80-100° C.to give a final concentration of 15% (w/v) glycerol.

The first and second pre-mixes were then combined by mixing equalamounts of each pre-mix to give the final concentrations of eachingredient, namely, the specified amount of the carbon particles, 15%(w/v) glycerol, 1% (w/v) Tween® 80, 1% (w/v) benzyl alcohol, 0.01% (w/v)simethicone.

The size of the carbon particle size in each final solution wasdetermined using a Nanotrac Wave II Laser Light Scattering Particle Sizeanalyzer without any further dilution or sonication or homogenization.

The darkness of the resulting solutions was measured by densitometryusing an x Rite 504 Portable Solar Reflection Spectrodensitometer.

The effect of carbon concentration as well as deagglomeration on thedepth color and the resulting % absorbed light is summarized below inTABLE 1, and shown pictorially in FIG. 2.

TABLE 1 Darkness % Carbon (% absorbance) - Particle non deagglomeratedDarkness (% absorbance) - Concentration (mean particle size aboutdeagglomerated (mean (w/v) 6.6 μm) particle size less than 0.2 μm) 0.025Too low to test 13.5%   0.05 Too low to test 31% 0.1 Too low to test 42%0.25 18%* 54% 0.50 27% 60% 1.0 43% 68% *The formulation containing 0.25%(w/v) carbon particles is representative of a commercially availablecarbon-based tissue staining solution containing carbon particles with amean particle diameter of about 6.6 μm.

As can be seen from TABLE 1 and FIG. 2, higher amounts of carbonparticles in a sample can result in darker marking solutions. However,the tissue staining compositions that had a mean particle diameter ofless than 0.2 μm were much darker than tissue staining compositionscontaining a mean particle diameter of about 6.6 μm. This experimentdemonstrates that a tissue staining composition containing carbonparticles at a concentration of 0.1% (w/v) and having a mean particlediameter less than 0.2 μm is about as dark as a tissue stainingcomposition containing carbon particles having a mean particle diameterof about 6.6 μm at a concentration of 1.0% (w/v), an order of magnitudegreater.

A solution containing only 0.025% (w/v) deagglomerated carbon particleshas a darkness equivalent to a solution containing 10 times more carbonby weight (0.25% (w/v), that had not been deagglomerated.

Example 2—Effects of Carbon Particle Size on Particle Sedimentation(Settling)

This example demonstrates that the size of the carbon particles can havea profound effect on the settling of the carbon particles.

In this example, two tissue stains were prepared that were the same(containing 1% (w/v) Tween® 80, 15% (w/v) glycerol, 1% (w/v) benzylalcohol, and 0.01% (w/v) simethicone), and containing 0.27% (w/v) carbonparticles (Monarch 4750 from Cabot Corp., Billerica Mass.) that wereeither not deagglomerated and thus contained carbon particles having amean particle diameter of about 6.6 μm or deagglomerated byhomogenization to produce carbon particles having a mean diameter lessthan 0.2 μm. The resulting particles were analyzed for particle size andstability.

The tissue stain containing the deagglomerated carbon particles wereprepared from a carbon particle pre-mix containing the ingredients setforth in TABLE 2A, and a Tween® 80 premix set forth in TABLE 2B.

TABLE 2A Carbon Particle Pre-Mix Ingredient Weight Tween ® 80 100 mLGlycerin 210 mL Simethicone 3.5 mL Benzyl alcohol 14 mL Carbon (Monarch4750, Cabot) 70 g SWFI 1,072 mL Total: 1,399.5 mL

In order to produce the carbon particle pre-mix, the Tween® 80,simethicone and glycerin were added to SWFI at 80° C. Once theingredients had dissolved, the resulting mixture was cooled to roomtemperature to produce a Tween® 80, simethicone, glycerin (TSG) mixture.One third of the resulting TSG mixture was combined with all the carbon(Cabot), and the carbon was wetted by stirring slowly until all the allthe carbon was wetted. The carbon containing mixture was homogenized for10 minutes at 4,000 rpm in a mixer (Silverson Model L5M-A) until all thecarbon was dispersed in solution without any large clumps. The resultingmixture was then added to another one third of the TSG mixture while themixer (Silverson Model L5M-A) was running at 1,000 rpm for 5-10 minutes.The remaining one third of the TSG mixture was added to the homogenizingmixture for an additional 45 minutes at 5,600 rpm. The benzyl alcoholwas then added for the last 1 minute of homogenization.

The carbon particle pre-mix can be used as is or stored until furtherprocessing. If the latter, the carbon particle pre-mix should behomogenized again for 10 minutes at 3,800 rpm (Silverson Model L5M-A)prior to further processing. In this example, the carbon particlecontaining pre-mix was homogenized for 10 minutes at 3,800 rpm in a(Silverson Model L5M-A) prior to the addition of further reagents, forexample, by combination with a Tween® 80 pre-mix, which was preparedcontaining the ingredients set forth in TABLE 2B.

TABLE 2B Tween ® 80 Pre-mix Ingredient Weight Tween80 ® 162 mL Glycerin3,690 mL   Simethicone 0 Benzyl alcohol 248 mL Cabot Carbon 0 SWFI19,600 mL   Premix & Total: 25,089.5 mL  

The Tween® 80 pre-mix was prepared as follows. 10 L of water waspreheated to a temperature in the range of 80° C.−100° C. In a separatecontainer, Tween® 80 was added to about 500 mL of SWFI at 80° C.−100° C.and mixed with stirring for 15 minutes. Then, the benzyl alcohol andglycerin are added to give a mixture, which is then added to the 10 L ofwater with mixing at 450 rpm. The remaining water was then added to formthe Tween® 80 pre-mix.

Thereafter, the carbon-particle pre-mix is then added to the Tween® 80pre-mix while mixing (ServoDyne Model SSM54) at 700 rpm for 15 minutesand then 450 rpm for 27 hours.

A similar tissue stain containing carbon particles that had not beendeagglomerated was produced as follows with the ingredients set forth inTABLE 2C.

TABLE 2C Ingredient Weight Tween ® 80 262 mL Glycerin 3,900 mLSimethicone 3.75 mL Benzyl alcohol 262 mL Cabot Carbon 68 g SWFI 20,745mL Premix & Total: 25,172.75 mL

A Tween® 80 pre-mix was prepared as follows: the Tween80® was added toabout 750 mL of SWFI at 80° C.-100° C. and mixed with stirring for 15minutes. A Simethicone pre-mix was prepared as follows: the simethiconewas added to 245 mL of SWFI and mixed for 15 minutes.

The glycerin was added to 10 L of SWFI at 80° C.-100° C. in a ServoDynemixer operating at 450 rpm and mixed for 10 minutes, and then theremainder of the SWFI was added. Thereafter, the Tween80® pre-mix wasadded and mixed for 10 minutes, the benzyl alcohol was then added andmixed for 5 minutes, and the simethicone pre-mix was then added andmixed for 5 minutes. Finally the carbon was added, and the resultingmixture mixed for about 20 hours.

Once prepared, the particle sizes of the carbon particles in each tissuestain were measured with a Nanotrac Wave II Laser Light ScatteringParticle Size analyzer. The carbon particles in the tissue staincontaining the deagglomerated carbon particles had a mean particle sizeof less than 0.2 μm (0.17 μm), whereas the carbon particles present inthe tissue stain that had not been deagglomerated had a mean particlediameter of about 6.6 μm.

In addition, each tissue stain was analyzed with a LUMiSizer 650 (LUMBmbH, Berlin, Germany) to determine stability (sedimentation) at roomtemperature. The sedimentation rates can be expressed as the mediansedimentation rate (μm/s) or the D90 sedimentation rate (μm/s; 90% ofall the particles have a sedimentation rate less than the D90 value,whereas 10% of the particles have a sedimentation rate higher than theD90 value).

It was found that the tissue stain containing the particles that werenot deagglomerated had a median sedimentation rate of about 8 μm/s and aD90 sedimentation rate of about 171 μm/s, whereas the tissue staincontaining the particles that were deagglomerated had a mediansedimentation rate of 2.7 μm/s and a D90 sedimentation rate of about 4.9μm/s.

Given the size of the deagglomerated particles in the tissue stain, itis contemplated that sedimentation will be reduced or even eliminateddue to Brownian back diffusion if the carbon particles are preventedfrom reagglomerating, for example, by the presence of an effectiveamount of a surfactant, for example, a non-ionic surfactant.

Example 3—Carbon Particle-Based Tissue Stains with DesirableAnti-Settling and Mucoadhesive Properties

This example demonstrates that it is possible to create a carbonparticle-based tissue staining composition that is more resistant tosettling than commercially available tissue staining solutions. The sizeof the carbon particles has a profound effect on the settling of theparticles. For example, carbon particles having a mean particle diameterless than 0.3 μm (for example, less than 0.2 μm) exhibit much lowersedimentation over time especially in the presence of an agent, forexample, a surfactant, such as a non-ionic surfactant, that preventsreagglomeration of the carbon particles.

However, under certain circumstances, it may be desirable to combine thecarbon particles (e.g., in the presence of a surfactant) with ananti-settling agent. This example also demonstrates that the particlesize and/or the settling agent can be used to create a tissue stainingcomposition that has the appropriate anti-settling properties.

In addition, if the exemplary tissue staining compositions havemucoadhesive properties, it is contemplated that the carbon particleswill not diffuse out of the tissue regions of interest that have beenpreviously marked, thereby permitting users to visualize those regionsmore clearly and for longer periods of time than when using stainingsolutions without the mucoadhesive properties.

In this example, various tissue staining compositions were created andanalyzed by centrifugation to mimic standing for approximately 7 months.Each composition contained 15% glycerol, 1% Tween® 80, 1% benzyl alcoholand 0.01% simethicone in water. However, each particular tissue stainingcomposition contained either 0.50% (w/v) (TABLE 3) or 0.25% (w/v) (TABLE4) carbon particles (Monarch 4750 from Cabot Corp., Billerica Mass.),which were either deagglomerated by sonication as described in Example 1to reduce particle size or not, together with different types andamounts of polymers (e.g., hydroxyethyl cellulose (HEC, includingHEC300, HEC2K, and HEC5K from Spectrum Chemical or Ashland; guar gum(from Spectrum Chemical, Ashland), and hydroxypropyl cellulose (HPC 4956from Spectrum Chemical, Ashland, or Fisher). Solutions lacking thepolymer are referred to as “No polymer—control” in TABLES 3 and 4.

The darkness of the final tissue staining compositions solutions weremeasured as described in Example 1, and the results expressed as %absorbance.

The settling properties of the carbon particles in each of the varioustissue staining compositions were compared by centrifugation. Inparticular, 5 mL of each sample was placed in a centrifuge tube in aThermoScientific Multifuge X1R centrifuge and spun at 5,000×g for 60minutes (representative of settling at about 7 months). This resulted inthe settling of a portion of the carbon particles to give a pellet ofsettled solids at the bottom of each centrifuge tube and a liquidsupernatant above the pellet. An aliquot from the one third of thesupernatant was then removed and analyzed by UV/visible spectrometry todetermine carbon concentration, which was then compared to the carbonconcentration in the sample prior to centrifugation. Samples with lesssettling have more carbon remaining in the top third of the liquid aftercentrifugation, resulting in a lower percent loss.

The mucoadhesive properties of the tissue staining compositions werealso assayed. Briefly, an agar plate comprising 2% porcine mucin and 2%agar was prepared by dissolving 20 grams of granulated agar and 20 gramsof porcine mucin in 1000 mL of distilled water by boiling for 20minutes. The molten mucin-agar mix was poured into a plastic containerwith dimensions of approximately 13 inches long, 10 inches wide, and 2inches deep. The mucin-agar mix was allowed to solidify at roomtemperature. Then 75 μL of each sample was applied as spots to thesurface of the test plate, then the plates were inclined at an angle of30 degrees relative to horizontal and the distance travelled down theplate was measured after one minute. Samples that traveled a shorterdistance are more mucoadhesive than samples that traveled a longerdistance.

The results are summarized in TABLES 3 and 4.

TABLE 3 0.50% (w/v) Carbon Particles Settling (% loss SampleDeagglomerated Darkness upon Mucoadhesion No. % Polymer (w/v) particles(Y/N) (% abs) centrifugation) (travel (cm)) 1 0.2% HEC 300 Y 71% 74%13.8 2 0.10% HEC 2K Y 68% 49% 13.5 3 0.10% HEC 2K N 22% 97% 13 4 0.15%HEC 2K Y 68% 31% 11 5 0.20% HEC 2K Y 61% 32% 10.3 6 0.20% HEC 2K N 26%90% 9.0 7 0.05% HEC 5K Y 70% 83% 17.5 8 0.10% HEC 5K Y 69% 53% 14 90.10% HEC 5K N 37% 90% 13 10 0.20% HEC 5K Y 61% 20% 8.8 11 0.20% HEC 5KN 21% 90% 7.5 12 0.15% Guar gum Y 67% 56% 12.3 13 0.30% Guar gum Y 61%58% 5 14 0.1% HPC 4956 Y 64.9%   80% 16.25 15 No polymer - Y 60% 85% 20control 16 No polymer - N 27% 98% 19.5 control

The results are also compiled as a bar chart in FIG. 3. The resultsdemonstrate (e.g., by comparing the results from samples 7, 10 and 11)that, in order to prevent settling of the particles over prolongedperiods of time, it is necessary to reduce the size of the carbonparticles (compare samples 10 and 11) and to include an appropriateamount of a polymer that demonstrates anti-settling properties (comparesamples 7 and 10). In particular, when the particles were deagglomeratedby sonication and present in a solution containing 0.2% HEC5K (sample10), less than 20% of the particles settled out under the conditionstested. However, when only 0.05% HEC5K (sample 7) was present in thesample, over 80% of the particles settled under the conditions tested,and when the particles were not deagglomerated but yet present in asolution containing 0.20% HEC5K (sample 10) approximately 90% of theparticles settled out under the conditions tested. Similar trends whereobserved when HEC2K was used instead of HEC5K.

With regard to mucoadhesion, it was found that the HEC2K and HEC5K, inaddition to preventing the settling of the particles, also weremucoadhesive. When the staining solution lacked a polymer, the solutionmigrated along the mucin/agar plates about 20 cm in 1 minute, whereasthe staining solution containing 0.20% HEC5K migrated about 8.8 cm in aminute.

All of the solutions (Sample Nos. 1-16) required less than 23N ofapplied pressure to pass the solution through a 25 gauge needle having alength of 240 cm (Interject, Boston Scientific, MA). As a result, it iscontemplated these test solutions can be delivered by such an injectionneedle to facilitate marking of gastrointestinal tissue.

TABLE 4 0.25% (w/v) Carbon Particles Deagglomerated Settling (% lossSample carbon particles Darkness upon Mucoadhesion No. % Polymer (w/v)(Y/N) (% abs) centrifugation) (travel (cm)) 17 0.10% HEC 2K Y 46% 81% 1318 0.10% HEC 2K N 18% 92% 13 19 0.20% HEC 2K Y 49% 11% 9 20 0.20% HEC 2KN 20% 86% 9 21 0.10% HEC 5K Y 60% 35% 12.5 22 0.10% HEC 5K N 22% 92%12.5 23 0.20% HEC 5K Y 47% 13% 7.5 24 0.20% HEC 5K N 16% 86% 7.5 25 Nopolymer - Y 54% 92% 20 control 26* No polymer - N 18% 98% 19.8 control*This formulation is representative of a commercially available carbonparticle-based tissue staining solution.

The results from TABLE 4 are also compiled as a bar chart in FIG. 4.

Similar results were obtained when the carbon particle concentration waslowered from 0.5% to 0.25% (w/v). For example, by comparing the resultsfrom samples 19 and 20 (both containing 0.2% HEC2K), when the carbonparticles were deagglomerated by sonication, only about 11% of theparticles settled out (sample 19) under the conditions tested, but whenthe carbon particles were not deagglomerated, about 90% of the particlessettled out (sample 20) under the conditions tested. Similar resultswere observed when HEC2K was replaced with HEC5K (see samples 23 and24).

The darkness of each of the solutions set forth in TABLES 3 and 4 weretested as described in Example 1. The results are summarized in FIGS. 5and 6. The results demonstrate that the effect of particle size in thepresence of an anti-settling agent (for example, HEC2K or HEC5K) canhave a profound impact on the darkness of the carbon particles. SeeFIGS. 5 and 6, which represent the darkness of solutions containing0.25% or 0.5% (w/v) carbon particles that had been sonicated (S) or notsonicated (NS) in the presence of HEC2K (FIG. 5) or HEC5K (FIG. 6). Theresults demonstrate that sonication (deagglomeration) of the carbonparticles has a profound effect on the darkness (expressed as %absorbance) of the staining solutions. Even in the presence of ASA, thesolutions containing carbon particles having a mean particle diameterless than 0.3 μm were much darker than comparable solutions where thecarbon particles were not deagglomerated by sonication and had a meanparticle diameter of about 6.6 μm.

With regard to mucoadhesion, it was found that the HEC2K and HEC5K, inaddition to preventing the settling of the particles, also weremucoadhesive. When the staining solution lacked a polymer (see, samples25 and 26), the solution migrated on the mucin/agar plates about 20 cmin 1 minute, whereas the staining solution containing 0.20% HEC5Kmigrated only about 8 cm in a minute.

The results are also shown in FIG. 7, which demonstrates that as theconcentration of the ASA with mucoadhesive properties (e.g., HEC2K orHEC5K) increased (for example, from 0.075% to 0.2% (w/v)), themucoadhesive properties of the tissue staining solution also increased,as expressed by the distance travelled over 1 minute. The higher theconcentration of the HEC, the shorter the distance travelled on theagar/mucin plates. These results are shown visually in FIG. 8, whichshow that HEC5K has mucoadhesive properties, and that the tissuestaining compositions visually look much darker to the eye when the sizeof the carbon particle agglomerates have been reduced by sonication.Staining solutions containing guar gum also were mucoadhesive.

The darkness and mucoadhesive properties are shown visually in FIGS. 8Aand 8B where the stains containing different concentrations of HEC5K inthe presence of 0.25% (w/v) carbon particles which are notdeagglomerated. In each of FIGS. 8A and 8B, samples 1, 2, 3, 4, 5, and 6correspond to respective staining solutions 26 (no HEC,non-deagglomerated carbon particles), 25 (no HEC, deagglomerated carbonparticles), 22 (0.1% HEC5K, non-deagglomerated carbon particles), 21(0.1% HEC5K, deagglomerated carbon particles), 24 (0.2% HEC5K,non-deagglomerated carbon particles), and 23 (0.2% HEC5K, deagglomeratedcarbon particles). The staining solutions containing the deagglomeratedcarbon particles (lines 2, 4, 6) were much darker than the stainingsolutions containing the non-deagglomerated carbon particles.Furthermore, as the concentration of HEC increased, the samples weremore mucoadhesive as demonstrated by the size of each spot before theplates were tilted (FIG. 8A) where the smaller spots were moremucoadhesive, and after the plates were tilted (FIG. 8B) where thesamples that travelled the shortest distance were the most mucoadhesive.

The exemplary stains described in this Example are much moremucoadhesive than comparable, surfactant containing, commerciallyavailable carbon particle-based tissue stains. As a result, it iscontemplated that the tissue stains of the invention will not diffuse orbleed out of the tissue regions of interest that have been previouslymarked, thereby permitting users to visualize those regions more clearlyand for longer periods of time than when using the prior art stain.

All of the solutions (Sample Nos. 17-26) required less than 23N ofapplied pressure to press the solution through a 25 gauge needle havinga length of 240 cm (Injector, Boston Scientific, MA). As a result, eachof the tissue stains in this example can be introduced into a tissue ofinterest using commercially available needles and/or injection systems.

INCORPORATION BY REFERENCE

The entire disclosure of each of the patent and scientific documentsreferred to herein is incorporated by reference for all purposes.

EQUIVALENTS

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

What is claimed is:
 1. A liquid tissue staining composition comprising:a thermally sterilized mixture comprising: deagglomerated carbonparticles at a concentration of from about 0.025% to 2.0% (w/v) having amean particle diameter from about 0.05 μm to less than 0.2 μm;hydroxyethyl cellulose; and at least one excipient selected from thegroup consisting of (a) a viscosity-increasing agent selected from thegroup consisting of glycerol, propylene glycol, isopropylene glycol,polyethylene glycol, and cellulose; (b) a mucoadhesive agent selectedfrom the group consisting of collagen, gelatin, alginate, chitosan,polylactic acid, polyglycolic acid, and any combination thereof, and (c)a surfactant selected from the group consisting of polyethoxylatedsorbitan ester, sorbitan ester, and any combination thereof wherein,when the liquid tissue staining composition is injected into asubmucosal layer, the resulting mark can be visualized after one month.2. The liquid tissue staining composition of claim 1, wherein thehydroxyethyl cellulose is included at a concentration of from about0.025% to less than 5.0% (w/v).
 3. The composition of claim 1, wherein,when centrifuged for 60 minutes at 5,000×g, less than 50% of the carbonparticles settle out of solution.
 4. The composition of claim 1, whereinthe excipient comprises the viscosity-increasing agent.
 5. Thecomposition of claim 4, wherein the viscosity-increasing agent ispresent at a concentration of from about 5% to about 25% (w/v).
 6. Thecomposition of claim 1, further comprising an anti-foaming agent.
 7. Thecomposition of claim 6, wherein the anti-foaming agent is present at aconcentration of from about 0.005% to about 1.0% (w/v).
 8. Thecomposition of claim 6, wherein the anti-foaming agent is selected fromthe group consisting of dimethicone and simethicone.
 9. The compositionof claim 1, wherein the surfactant is present at a concentration of fromabout 0.01% to about 2.0% (w/v).
 10. The composition of claim 1, whereinthe surfactant comprises polyoxyethylene sorbitan esterified with fattyacid.
 11. The composition according to claim 1, wherein the carbonparticles are derived from carbon black, activated carbon, unactivatedcarbon or a combination thereof.
 12. The composition of claim 1, whereinthe carbon particles have a level of polycyclic aromatic hydrocarbons ofno greater than 0.5 ppm based on the total amount of carbon particles.13. The composition of claim 1, wherein the composition is terminallysterilized.
 14. The composition of claim 1, wherein the composition iscapable of passing through a 25 gauge needle 240 cm in length byapplication of a force no greater than about 32N to push the compositionthrough the needle.
 15. The composition of claim 1, wherein thecomposition further comprises an anti-settling agent (ASA), and theconcentration of the ASA is from about 0.05% (w/v) to about 1% (w/v).16. The composition of claim 6, comprising either: (a) from about 0.025%to 2.0% (w/v) of the carbon particles; from less than 0.025% to about5.0% (w/v) of the hydroxyethyl cellulose; when present, from about 5.0%to about 25% (w/v) of the viscosity-increasing agent; from about 0.005%to about 1.0% (w/v) of the anti-foaming agent; and from about 0.1% toabout 2.0% (w/v) of the surfactant; or (b) from about 0.025% to about1.0% (w/v) of the carbon particles; from about 0.25% to about 1.0% (w/v)of the hydroxyethyl cellulose; when present, from about 12% to about 18%(w/v) of the viscosity-increasing agent; from about 0.05% to about 0.25%(w/v) of the anti-foaming agent; and from about 0.7% to about 1.5% (w/v)of the surfactant.
 17. The composition of claim 1 further comprising apreservative.
 18. The composition of claim 17, wherein the preservativeis benzyl alcohol.
 19. A method of preparing the tissue stainingcomposition of claim 1, the method comprising: (a) providing acomposition comprising deagglomerated carbon particles having a meanparticle diameter from about 0.05 μm to less than 0.2 μm and optionallya surfactant; and (b) combining the composition comprising the carbonparticles with the hydroxyethyl cellulose and an optional mucoadhesiveagent or viscosity increasing agent to produce the tissue stainingcomposition.
 20. The method of claim 19, wherein the carbon particles instep (a) are produced by sonicating or homogenizing carbon particleshaving mean particle diameter of greater than 5 μm in diameter.
 21. Themethod of claim 19, wherein, during step (a), the composition comprisesthe surfactant and further comprises an anti-foaming agent.
 22. A methodof staining a region of tissue in a subject, the method comprisinginjecting the tissue staining composition of claim 1 into the region ofinterest in the subject in an amount effective to stain the region so asto be visible by visual inspection.
 23. The method of claim 22, whereinthe tissue is tissue present in the gastrointestinal tract, bladder,lung, breast, lymph node, or central or peripheral nervous system of thesubject.
 24. The composition of claim 16 comprising from about 0.1% to2% (w/v) carbon particles.
 25. The composition of claim 16, wherein thecomposition comprises the viscosity-increasing agent.
 26. A liquidtissue staining composition consisting of: a thermally sterilizablemixture consisting of deagglomerated carbon black-derived carbonparticles at a concentration of from about 0.1% to about 0.5% (w/v)having a mean particle diameter from about 0.05 μm to about 0.2 μm; aviscosity-increasing agent selected from the group consisting ofglycerol, propylene glycol, isopropylene glycol, polyethylene glycol,cellulose, and any combination thereof present at a concentration offrom about 10% to about 20% (w/v); a surfactant comprisingpolyoxyethylene sorbitan esterified with fatty acid present at aconcentration of about 0.5% to about 2.0% (w/v); an antifoaming agentselected from the group consisting of dimethicone, simethicone, and anycombination thereof present at a concentration of from about 0.01% toabout 0.5% (w/v); a preservative selected from the group consisting ofbenzyl alcohol, methyl paraben, ethyl paraben, benzalkonium chloride,and any combination thereof; and, water, wherein, when the liquid tissuestaining composition is injected into a submucosal layer, the resultingmark can be visualized after one month.
 27. The liquid tissue stainingcomposition of claim 26, wherein the preservative is benzyl alcohol andis present in an amount of from about 0.01% to about 4.0% (w/v).
 28. Thecomposition of claim 16, comprising from about 0.1% to about 1.0% (w/v)of the carbon particles.
 29. The composition of claim 1, wherein theexcipient comprises a surfactant present in an amount of from about 0.5%to about 1.5% (w/v).
 30. A pre-filled syringe, the pre-filled syringefilled with the composition according to claim
 1. 31. The pre-filledsyringe of claim 30, wherein the thermally sterilized mixture issterilized by autoclaving the pre-filled syringe.